Conformable tissue repair implant capable of injection delivery

ABSTRACT

A conformable tissue implant is provided for use in repairing or augmenting a tissue defect or injury site. The tissue implant contains a tissue carrier matrix comprising a plurality of biocompatible, bioresorbable granules and at least one tissue fragment in association with the granules. The tissue fragment contains one or more viable cells that can migrate from the tissue and populate the tissue carrier matrix. Also provided is a method for injectably delivering the tissue implant.

CROSS REFERENCE TO RELATED APPLICATIONS

The present invention is a continuation of U.S. patent application Ser.No. 11/947,384, filed on Nov. 29, 2007, entitled “Conformable TissueRepair Implant Capable of Injection Delivery,” now U.S. Pat. No.7,875,296, and a continuation of U.S. patent application Ser. No.10/723,982, filed on Nov. 26, 2003, entitled “Conformable Tissue RepairImplant Capable of Injection Delivery,” now U.S. Pat. No. 7,316,822, theentire contents of which are hereby incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Not applicable.

FIELD OF THE INVENTION

The present invention relates to methods and apparatus for the treatmentof tissue injuries or defects. Specifically, the present inventionrelates to tissue repair and augmentation implants, and moreparticularly, to a conformable tissue repair and augmentation implantcapable of injection and a method for its minimally invasive delivery.

BACKGROUND OF THE INVENTION

Injuries to soft tissue, such as cartilage, skin, muscle, bone, tendonand ligament, where the tissue has been injured or traumatizedfrequently require surgical intervention to repair the damage andfacilitate healing. Such surgical repairs can include suturing orotherwise repairing the damaged tissue with known medical devices,augmenting the damaged tissue with other tissue, using an implant, agraft or any combination of these techniques. Despite these conventionalmethods of tissue repair, there is a continuing need in this art fornovel surgical techniques for the surgical treatment of damaged tissue(e.g., cartilage, meniscal cartilage, ligaments, tendons and skin) thatcan effect a more reliable tissue repair over the long term and canfacilitate the healing of injured tissue.

Recently, tissue engineering approaches to repairing tissue damage orinjury have been used with increasing frequency. These methods typicallyinvolve replacing or reconstructing damaged or injured tissue with cellscapable of new tissue growth. The cells are usually incorporated into adelivery vehicle such as a surgical implant for placement at the tissuesite, whereupon the healthy cells can grow into their surroundingenvironment. Various surgical implants are known and have been used insurgical procedures to help achieve these benefits. For example, it isknown to use various devices and techniques for creating implants havingisolated cells loaded onto a delivery vehicle. Such cell-seeded implantsare used in an in vitro method of making and/or repairing cartilage bygrowing cartilaginous structures that consist of chondrocytes seededonto biodegradable, biocompatible fibrous polymeric matrices. Suchmethods require the initial isolation of chondrocytes from cartilaginoustissue prior to the chondrocytes being seeded onto the polymericmatrices. Other techniques for repairing damaged tissue employ implantshaving stem or progenitor cells that are used to produce the desiredtissue. For example, it is known to use stem or progenitor cells, suchas the cells within fatty tissue, muscle, or bone marrow, to regeneratebone and/or cartilage in animal models. The stem cells are removed fromthe animal and placed in an environment favorable to cartilageformation, thereby inducing the fatty tissue cells to proliferate and tocreate a different type of cell, such as cartilage cells.

While the trend towards using tissue engineering approaches to tissuerepair continues to gain popularity, mainly because of the long-termbenefits provided to the patient, these current techniques are notwithout drawbacks. One disadvantage with current tissue engineeringtechniques is that they can be time consuming. A typical processinvolves the harvest of cellular tissue in a first surgical procedure,which is then transported to a laboratory for cell culturing andamplification. The tissue sample is treated with enzymes that willrelease the cells from the matrix, and the isolated cells will be grownfor a period of 3 to 4 weeks using standard cell culture techniques.Once the cell population has reached a target number, the cells are sentback to the surgeon for implantation during a second surgical procedure.This manual labor-intense process is extremely costly and timeconsuming. Although the clinical data suggest long term benefits for thepatient, the prohibitive cost of the procedure combined with thetraumatic impact of two surgical procedures, has hampered adoption ofthis technique.

The current model for tissue repair generally involves retrieving a cellsample from a patient, isolating the cells, culturing the cells forseveral weeks, and then implanting them in a defect, either with orwithout a scaffold. Preferably, a scaffold is used in order tofacilitate newly developing cell growth. In the past, such scaffoldshave consisted mostly of two- or three-dimensional porous scaffolds thatallow cell invasion and remodeling once the scaffold has been combinedwith living cells and has been delivered inside the patient. This modelis limited in application because of the secondary surgery and highcosts involved. More importantly, one limitation of using such scaffoldsis that tissue defect geometry can often be unpredictable. Since thescaffold geometry is essentially limited to what has been manufactured,the scaffold carrier to be implanted rarely matches perfectly the site.In order to achieve a desirable complementary fit with the defect orinjury site, the scaffold often needs to be revised by trimming prior toor after implantation. This additional adjustment time adds onto theoverall surgery time for the patient. For certain difficult to match orunusually shaped sites, even the step of trimming the scaffold does notensure an ideal fit with the implantation site. Further, whererelatively large tissue defects are involved, minimally invasive surgerymay not be possible due to the limited size of the surgical access site.Therefore, delivery of large scaffolds may require an open procedurewhich poses more risks to the patient.

Injectable gels and microcarrier beads have also been used in the pastas cell delivery vehicles. These systems have the advantage of sometimesbeing injectable and therefore require less invasive procedures forimplantation. Typically, these carriers have been combined with isolatedcells, which are sensitive to manipulation such as shear, or thepresence of crosslinkers that are required to allow the carrier to befixed or set in place. Hence, these systems have proven to be less thanideal due to the problems associated with cell viability onceincorporated into these carrier systems. Accordingly, there continues toexist a need in this art for a method of delivering tissue repairimplants through a minimally invasive procedure. Also desirable is aconformable tissue repair or augmentation implant that can adapt to theshape or geometry of the tissue site. The implant should be suitable fordelivering viable tissue capable of effecting new cell growth. It isalso desirable to provide a method for making such an implant, wherebythe implant can be made in a quick and efficient manner for immediateuse during surgery.

SUMMARY OF THE INVENTION

This invention relates to a conformable tissue implant for use intreating injured or defective tissue, and a method for delivering suchan implant in a minimally invasive procedure. The implant is configuredto be introduced to the tissue site, where it can assume the shape orgeometry of the tissue defect or injury site, thereby providing a closeinterface between the implant and the tissue site which enhances healingand promotes new cellular growth to occur. The biocompatible tissueimplant can be used for the repair, augmentation and/or regeneration ofdiseased or damaged tissue. Further, the tissue implant can be used fortissue bulking, cosmetic treatments, therapeutic treatments, tissueaugmentation, as well as tissue repair.

The tissue repair implant of the present invention comprises finelyminced tissue fragments combined with a tissue carrier matrix formed ofbiocompatible, bioresorbable granules. The tissue fragments can bederived from a number of sources, including connective tissue such ascartilage, meniscus, tendon, ligament, dermis, bone, or combinationsthereof. In addition, the tissue fragments can be autogenic tissue,allogeneic tissue, xenogeneic tissue, or combinations thereof. Thetissue fragments serve as a cell source for new cellular growth, andhave an effective amount of viable cells that can migrate out of thetissue fragment and populate the tissue carrier matrix once the implantis delivered to the patient. The granules serve as a microcarrier toprovide sufficient mechanical integrity for cellular integration withthe surrounding environment during the tissue remodeling process. In oneaspect of the present invention, the finely minced tissue fragments andgranules together form an injectable suspension that can be delivered byinjection in a minimally invasive procedure. Over time, the plurality ofbiocompatible, bioresorbable granules are resorbed to leave behind thenew tissue at the implant site.

In one embodiment of the present invention, the tissue carrier matrixfurther includes a binding agent that acts to gel together or facilitatecohesion of the tissue fragments and granules within the tissue carriermatrix. The binding agent enables the implant to take on a semi-solid orgel-like form. Where a solid or cured implant is desired, a curing agentcan additionally be provided with the tissue carrier matrix. This curingagent would act to crosslink the binding agent, thereby forming a solidimplant within which are the tissue fragments and the bioresorbable,biocompatible granules. In one aspect, the implant is cured once it isdelivered to the implantation site. In another aspect, the implant iscured prior to its delivery to the implantation site. To further enhancethe implant's regenerative or reconstructive abilities, the tissuecarrier matrix can also include a biological component or effector whichenhances the effectiveness of the tissue fragments to new cellulargrowth.

The invention also provides to a method of repairing a tissue defect orinjury which involves the steps of providing a tissue repair implant inaccordance with the present invention and delivering the tissue repairimplant to a tissue defect or injury site. In one aspect, the step ofdelivering includes injecting the tissue repair implant into the tissuedefect site. The tissue carrier matrix can also include a curing agent,and the method of the present invention can further include the step ofallowing the tissue repair implant to set at the tissue defect site.Alternatively, the tissue repair implant can be allowed to set prior todelivering the tissue repair implant to the tissue defect or injurysite. At least one tissue fragment associated with the tissue carriermatrix comprises a type that is the same as the tissue to be treated.However, the tissue fragment can also comprise a type that is differentfrom the tissue to be treated.

The invention also provides a method of preparing a tissue repairimplant in accordance with the present invention, which involves thesteps of providing a tissue carrier matrix comprising a plurality ofbiocompatible, bioresorbable granules, introducing a fluid suspensioncontaining at least one tissue fragment to the tissue carrier matrix,the tissue fragment having an effective amount of viable cells capableof migrating out of the tissue fragment and into the tissue carriermatrix, separating the at least one tissue fragment from the fluidsuspension, and collecting the tissue carrier matrix with the at leastone tissue fragment for implantation at a tissue site to be repaired.The tissue carrier matrix can be provided with a binding agent whichenables the implant to form a gel-like or semi-solid implant. A curingagent can additionally be provided to enable the implant to set eitherbefore or after delivery to the implantation site. A biologicalcomponent or an effector can also be added to the tissue carrier matrixto further enhance the effectiveness of the tissue fragments.

The tissue implant of the present invention aims to accomplish severaltasks simultaneously in order to provide more efficient delivery of atissue repair implant to a site of tissue injury or defect. Theinvention combines the utility of sieving or capturing a biologicalagent in a carrier, with the advantage of being able to immediately usethe biological agent in an intraoperative procedure, in order to delivera conformable tissue implant loaded with tissue fragments containingliving cells to a tissue implantation site. Another advantage providedby the tissue repair implant of the present invention is that there isno need to isolate cells, nor is there a need to grow tissue or attachcells to the carrier prior to delivering the implant to the implantationsite. Also, by using a carrier comprising bioabsorbable, biocompatiblegranules, the implant is able to combine sustained drug deliverycapabilities and structural integrity provided by a scaffold supportwith the convenience of injection delivery.

In embodiments in which the implant is used for tissue repair, thetissue repair implant can be used to treat a variety of injuries, suchas injuries occurring within the musculoskeletal system (e.g., rotatorcuff injuries, ACL ruptures, and meniscal tears), as well as injuriesoccurring in other connective tissues, such as skin and cartilage.Furthermore, such implants can be used in other orthopedic surgicalprocedures, such as hand and foot surgery, to repair tissues such asligaments, nerves, and tendons.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a photomicrograph showing a section of one embodiment of thetissue repair implant which comprises cartilage fragments, 158 μm PGAgranules, and fibrin glue in accordance with the present invention.

FIG. 1B is a photomicrograph of a histological section of an implantsimilar to FIG. 1A, after 3 weeks in vitro.

FIG. 1C is a photomicrograph of a histological section of an implantsimilar to FIG. 1A, after 6 weeks in vitro.

FIG. 1D is yet another photomicrograph of a histological section of theimplant of FIG. 1C.

FIG. 2A is a photomicrograph of a histological section of anotherembodiment of the tissue repair implant comprising cartilage fragments,286 μm PGA granules, and fibrin glue in accordance with the presentinvention.

FIG. 2B is a photomicrograph of a histological section of an implantsimilar to FIG. 2A, after 3 weeks in vitro.

FIG. 2C is a photomicrograph of a histological section of an implantsimilar to FIG. 2A, after 6 weeks in vitro.

FIG. 2D is yet another photomicrograph of a histological section of theimplant of FIG. 2C.

FIG. 3A is a photomicrograph of a histological section at 100×magnification of yet another embodiment of a tissue repair implantcomprising cartilage fragments, 633 μm PLA granules and fibrin glue,after 3 weeks in vitro.

FIG. 3B is a photomicrograph of a histological section at 100×magnification of a tissue repair implant similar to FIG. 3A, after 6weeks in vitro.

FIG. 4A is a photomicrograph of a histological section at 100×magnification of a tissue repair implant comprising cartilage fragmentsand fibrin glue, after 3 weeks in vitro.

FIG. 4B is a photomicrograph of a histological section at 100×magnification of a tissue repair implant similar to FIG. 4A, after 6weeks in vitro.

DETAILED DESCRIPTION OF THE INVENTION

In general, the invention relates to a tissue repair implant thatcomprises finely minced tissue fragments combined with a tissue carriermatrix formed of a plurality of granules. The tissue fragments serve asa cell source for new cellular growth, and the tissue fragments have aneffective amount of viable cells that can migrate out of the tissuefragment to populate the tissue carrier matrix once the implant isdelivered to the patient. The granules serve as a microcarrier toprovide sufficient mechanical integrity for cellular integration withthe surrounding environment during the tissue remodeling process. In oneaspect of the present invention, the finely minced tissue fragments andgranules together form an injectable suspension that can be delivered byinjection to a target site in a minimally invasive procedure. Byproviding the implant in a suspension form, the implant is able toconform to any defect size, shape or geometry, and can assume a shapecomplementary to that of the implantation site. Ultimately, this featureof the invention provides an implant having a close interface with thetissue area to be repaired, thereby enhancing tissue remodeling andhealing.

The biocompatible tissue implants of the present invention are used inthe treatment of various types of tissue for various purposes. Forexample, the implants can be used for the remodeling, repair and/orregeneration of diseased or damaged tissue. Although the implants aresometimes referred to herein as “tissue repair implants” and the methodsof using the implants are sometimes characterized as tissue repairtechniques, it is understood that the implants can be used for a varietyof tissue treatments, including but not limited to tissue remodeling,tissue repair, tissue bulking, tissue augmentation, cosmetic treatments,therapeutic treatments, and for tissue sealing.

The tissue repair implant includes at least one sample of viable tissuethat is associated with at least a portion of the tissue carrier matrix.The term “viable,” as used herein, refers to a tissue sample having oneor more viable cells. Virtually any type of tissue can be used toconstruct the tissue repair implants of the present invention. Forexample, the tissue used can be obtained from a connective tissue suchas cartilage tissue, meniscal tissue, ligament tissue, tendon tissue,skin tissue, bone tissue, muscle tissue, periosteal tissue, pericardialtissue, synovial tissue, nerve tissue, fat tissue, kidney tissue, bonemarrow, liver tissue, bladder tissue, pancreas tissue, spleen tissue,intervertebral disc tissue, embryonic tissue, periodontal tissue,vascular tissue, blood and combinations thereof. In one embodimentuseful for cartilage repair, the tissue is free of bone tissue and isselected from the group consisting of fibrocartilage tissue containingchondrocytes, meniscal tissue, ligament tissue and tendon tissue. Thetissue used to construct the tissue implant can be autogenic tissue,allogeneic tissue, or xenogeneic tissue. For example, healthy cartilagetissue, bone marrow tissue or aspirates are suitable for use with tissuerepair implants for repairing condylar surfaces. It is also contemplatedthat the tissue to be used can be of the same type or a different typethan the tissue to be treated with the implant.

In one embodiment useful for meniscal repair, the tissue used in thetissue repair implant can be selected from the group consisting ofmeniscal tissue, cartilage tissue, skin, synovial tissue, periostealtissue, pericardial tissue, fat tissue, bone marrow, blood, tendontissue, ligament tissue, or combinations thereof. The tissue can beobtained using any of a variety of conventional techniques, such as forexample, by biopsy or other surgical removal. Preferably, the tissuesample is obtained under aseptic conditions. Once a sample of livingtissue has been obtained, the sample can then be processed under sterileconditions to create a suspension having at least one minced, or finelydivided, tissue particle. The particle size of each tissue fragment canvary, for example, the tissue size can be in the range of about 0.1 toabout 3 mm³, in the range of about 0.5 to about 1 mm³, in the range ofabout 1 to about 2 mm³, or in the range of about 2 to about 3 mm³, butpreferably the tissue particle is less than about 1 mm³.

Preferably, the minced tissue fragment has at least one viable cell thatcan migrate from the tissue fragment into the tissue carrier matrix.More preferably, the tissue contains an effective amount of cells thatcan migrate from the tissue fragment and begin populating the tissuecarrier matrix of granules after implantation. In an optionalembodiment, the minced tissue fragments may be contacted with amatrix-digesting enzyme to facilitate cell migration out of theextracellular matrix surrounding the cells. The enzymes are used toincrease the rate of cell migration out of the extracellular matrix andinto the tissue carrier matrix. Suitable digesting enzymes that can beused in the present invention include, but are not limited to,collagenase, metalloproteinase, chondroitinase, trypsin, elastase,hyaluronidase, peptidase, dispase, thermolysin and protease.

In one embodiment, the minced tissue particles can be formed as asuspension in which the tissue particles are associated with aphysiological buffering solution. Suitable physiological bufferingsolutions include, but are not limited to, saline, phosphate buffersolution, Hank's balanced salts, Tris buffered saline, Hepes bufferedsaline and combinations thereof. In addition, the tissue can be mincedin any standard cell culture medium known to those skilled in the art,either in the presence or absence of serum. Prior to combining theminced tissue fragments with the granules of the tissue carrier matrix,the minced tissue suspension can be filtered and concentrated, such thatonly a small quantity of physiological buffering solution remains in thesuspension to prevent the tissue particles from drying out. Preferably,the minced tissue fragments in solution are at concentration in therange of approximately 1 to about 100 mg/cm², and more preferably in therange of about 1 to about 20 mg/cm².

The tissue samples used in the present invention are obtained from adonor (autogenic, allogeneic, or xenogeneic) using appropriateharvesting tools. The tissue samples can be finely minced and dividedinto small particles either as the tissue is collected, oralternatively, the tissue sample can be minced after it is harvested andcollected outside the body. In embodiments where the tissue sample isminced after it is harvested, the tissue samples can be weighed and thenwashed three times in phosphate buffered saline. Approximately 300 to500 mg of tissue can then be minced in the presence of a small quantity,such as, for example, about 1 ml, of a physiological buffering solution,such as, for example, phosphate buffered saline, or a matrix digestingenzyme, such as, for example, 0.2% collagenase in Hams F12. The mincingaction divides the tissue sample into particles or small pieces ofapproximately 1 mm³. Mincing the tissue can be accomplished by a varietyof methods. In one embodiment, the mincing is accomplished with twosterile scalpels using a parallel direction, and in another embodiment,the tissue can be minced by a processing tool that automatically dividesthe tissue into particles of a desired size. In another embodiment, theminced tissue can be separated from the physiological fluid andconcentrated using any of a variety of methods known to those havingordinary skill in the art, such as for example, sieving, sedimenting orcentrifuging with the bed of granules. In embodiments where the mincedtissue is filtered and concentrated, the suspension of minced tissuepreferably retains a small quantity of fluid in the suspension toprevent the tissue from drying out.

In the present invention, the minced tissue fragments are combined witha tissue carrier matrix formed of a plurality of granules. Preferably,the granules are formed from a bioresorbable or bioabsorbable materialthat has the ability to resorb in a timely fashion in the body. Overtime, the biocompatible, bioresorbable granules are resorbed to leavebehind the new tissue at the implant site. The granules can be formedfrom a variety of biocompatible, bioresorbable materials. For example,the granules can be formed from aliphatic polyesters,copoly(ether-esters), solid copolymers of fatty acid esters of glyceroland succinic acid, polyoxaesters, collagen, gelatin, albumin,hyaluronate, glucosaminoglycans, polyanhydrides, polyphosphazines,subintestinal mucosa, acellular tissues, and combinations thereof. Inaddition, the granules can be porous and/or have surface features suchas roughness or texture. Such features would further enhance theeffectiveness of the granules to attach and combine with the mincedtissue fragments as well as to the tissue implant site.

Suitable aliphatic polyesters include homopolymers or copolymers oflactides, glycolides, ε-caprolactone, p-dioxanone (1,4-dioxan-2-one),trimethylene carbonate (1,3-dioxan-2-one), and combinations thereof. Oneskilled in the art will appreciate that the differences in theabsorption time under in vivo conditions can be the basis for combiningtwo different polymers to form the granules of the present invention.For example, a copolymer of 35:65ε-caprolactone and glycolide (arelatively fast absorbing polymer) can be blended with40:60ε-caprolactone and L-lactide copolymer (a relatively slow absorbingpolymer) to form a suitable tissue carrier matrix.

Other useful polymers include polyphosphazenes, co-, ter- and higherorder mixed monomer based polymers made from L-lactide, D,L-lactide,lactic acid, glycolide, glycolic acid, para-dioxanone, trimethylenecarbonate and εε-caprolactone such as are described by Allcock in TheEncyclopedia of Polymer Science, Vol. 13, pages 31-41, WileyIntersciences, John Wiley & Sons, 1988 and by Vandorpe, et al in theHandbook of Biodegradable Polymers, edited by Domb, et al., HardwoodAcademic Press, pp. 161-182 (1997).

As used herein, the term “glycolide” is understood to includepolyglycolic acid. Further, the term “lactide” is understood to includeL-lactide, D-lactide, blends thereof, and lactic acid polymers andcopolymers.

Elastomeric copolymers are also particularly useful in the presentinvention. Suitable elastomeric polymers include those with an inherentviscosity in the range of about 1.2 dL/g to 4 dL/g, more preferablyabout 1.2 dL/g to 2 dL/g and most preferably about 1.4 dL/g to 2 dL/g asdetermined at 25° C. in a 0.1 gram per deciliter (g/dL) solution ofpolymer in hexafluoroisopropanol (HFIP). Further, suitable elastomersexhibit a high percent elongation and a low modulus, while possessinggood tensile strength and good recovery characteristics. In thepreferred embodiments of this invention, the elastomer exhibits apercent elongation greater than about 200 percent and preferably greaterthan about 500 percent. In addition to these elongation and modulusproperties, suitable elastomers should also have a tensile strengthgreater than about 500 psi, preferably greater than about 1,000 psi, anda tear strength of greater than about 50 lbs/inch, preferably greaterthan about 80 lbs/inch.

Exemplary biocompatible elastomers that can be used in the presentinvention include, but are not limited to, elastomeric copolymers ofε-caprolactone and glycolide (including polyglycolic acid) with a moleratio of ε-caprolactone to glycolide of from about 35:65 to about 65:35,more preferably from 45:55 to 35:65; elastomeric copolymers ofε-caprolactone and lactide (including L-lactide, D-lactide, blendsthereof, and lactic acid polymers and copolymers) where the mole ratioof ε-caprolactone to lactide is from about 35:65 to about 65:35 and morepreferably from 45:55 to 30:70 or from about 95:5 to about 85:15;elastomeric copolymers of p-dioxanone (1,4-dioxan-2-one) and lactide(including L-lactide, D-lactide, blends thereof, and lactic acidpolymers and copolymers) where the mole ratio of p-dioxanone to lactideis from about 40:60 to about 60:40; elastomeric copolymers ofε-caprolactone and p-dioxanone where the mole ratio of ε-caprolactone top-dioxanone is from about from 30:70 to about 70:30; elastomericcopolymers of p-dioxanone and trimethylene carbonate where the moleratio of p-dioxanone to trimethylene carbonate is from about 30:70 toabout 70:30; elastomeric copolymers of trimethylene carbonate andglycolide (including polyglycolic acid) where the mole ratio oftrimethylene carbonate to glycolide is from about 30:70 to about 70:30;elastomeric copolymers of trimethylene carbonate and lactide (includingL-lactide, D-lactide, blends thereof, and lactic acid polymers andcopolymers) where the mole ratio of trimethylene carbonate to lactide isfrom about 30:70 to about 70:30; and blends thereof. Examples ofsuitable biocompatible elastomers are described in U.S. Pat. No.5,468,253.

To form the granules, the biocompatible, bioresorbable polymer orcopolymer material is milled to a powder and the particles that areproduced serve as the granules. Once milled, the particles or granulescan be sieved and sorted by size. An appropriate range of sizes for thegranules of the present invention are in the range of about 150 μm toabout 600 μm in diameter. As explained in greater detail below, thegranules can have an average outer diameter in the range of about 150 to600 μm, and preferably in the range of about 150 to 300 μm. A bed ofthese beads or granules can be used to effectively sieve minced tissuefragments from a liquid suspension. The granules with the tissuefragments form a suspension that can be collected and loaded into aninjection device for delivery to an injury or diseased tissue site. Thegranules act as a carrier and also as a scaffold to support new tissuegrowth. Such a composition can conform to any defect geometry, enablingthe implant to assume a shape complementary to that of the implantationsite and provide enhanced healing.

In another embodiment of the present invention, the tissue carriermatrix further includes a binding agent that acts to gel together orfacilitate cohesion of the tissue fragments and granules, therebycreating a cohesive matrix. The binding agent enables the implant totake on a semi-solid or gel-like form which helps the suspension retaina given geometry while tissue remodeling occurs. For instance, thebinding agent could be a gel or biological or synthetic hydrogel so thatthe implant takes the form of an injectable gel. Suitable materials forthe binding agent include shark cartilage, alginate, hyaluronic acid,collagen gel, fibrin glue, fibrin clot, poly(N-isopropylacrylamide),agarose, chitin, chitosan, cellulose, polysaccharides,poly(oxyalkylene), a copolymer of poly(ethylene oxide)-poly(propyleneoxide), poly(vinyl alcohol), polyacrylate, platelet rich plasma (PRP)clot, platelet poor plasma (PPP) clot, Matrigel, blood clot,gelatin-resorcin-formalin adhesives, mussel-based adhesives,dihydroxyphenylalanine (DOPA) based adhesives, transglutaminase,poly(amino acid)-based adhesives, cellulose-based adhesives,polysaccharide-based adhesives, synthetic acrylate-based adhesives,liquid and semi-solid fatty acid esters of glycerol and succinic acid(MGSA), MGSA/polyethylene glycol (MGSA/PEG) copolymers,polyvinylpyrolidone (PVP), PVP copolymers, gelatin, albumin,monoglycerides, diglycerides, triglycerides laminin, elastin,proteoglycans, and combinations thereof.

Where a solid or cured implant is desired, a curing agent canadditionally be provided with the tissue carrier matrix to allow theinjectable implant to set in place at the defect site. This curing agentwould act to crosslink the binding agent, thereby forming a solidimplant within which are the tissue fragments and the bioresorbable,biocompatible granules. In one aspect, the implant is cured once it isdelivered to the implantation site. It is contemplated, however, thatthe implant can be allowed to cure prior to implantation as well, if sodesired. The curing agent should be selected so as to effectcrosslinking of the particular binding agent contained in the implant.Suitable curing agents include, for example, proteases such as thrombin,calcium, divinyl sulfone (DVS), polyethylene glycol divinyl sulfone(VS-PEG-VS), hydroxyethyl methacrylate divinyl sulfone (HEMA-DIS-HEMA),formaldehyde, glutaraldehyde, aldehydes, isocyanates, alkyl and arylhalides, imidoesters, N-substituted maleimides, acylating compounds,carbodiimide, hydroxychloride, N-hydroxysuccinimide, light (e.g., bluelight and UV light), pH, temperature, metal ions, and combinationsthereof. The present invention contemplates that, by using minced tissuefragments or particles rather than isolated cells, the naturalenvironment of the tissue fragments will provide sufficient protectionof the cells against the harsh reagents used for setting the implant.

To further enhance the implant's regenerative or reconstructiveabilities, the tissue carrier matrix can also include a biologicalcomponent such as an effector which enhances the effectiveness of thetissue fragments and facilitates tissue repair and healing of theinjured tissue. For example, in yet another embodiment of the presentinvention, the granules can be formulated to contain an effectivemolecule that would enhance the activity of the tissue fragments. Thus,the granules function in multiple ways by sieving the tissue fragments,providing a support and carrier for injection delivery to the site,providing a structural support for tissue remodeling, and potentiallydelivering other enhancing drug therapeutics to the site.

The biological component can be combined with the tissue carrier matrixin a variety of ways. For example, the biological component can becontained inside the granules themselves. For instance, the granules canbe porous to allow the biological component to be contained inside thepores. Alternatively, the biological component can be contained in aslow-release coating covering the granules. One skilled in the art willrecognize that the biological component can be incorporated into thegranules by any suitable manner known in the art that allows thegranules to administer the biological component to the minced tissuefragments, without affecting the effectiveness of the biologicalcomponent.

The biological component can be selected from among a variety ofeffectors that, when present at the site of injury, promotes healingand/or regeneration of the affected tissue. In addition to beingcompounds or agents that actually promote or expedite healing, theeffectors may also include compounds or agents that prevent infection(e.g., antimicrobial agents and antibiotics), compounds or agents thatreduce inflammation (e.g., anti-inflammatory agents), compounds thatprevent or minimize adhesion formation, such as oxidized regeneratedcellulose (e.g., INTERCEED and Surgicel®, available from Ethicon, Inc.),hyaluronic acid, and compounds or agents that suppress the immune system(e.g., immunosuppressants).

By way of example, other types of effectors suitable for use with theimplant of the present invention include antibiotics, antimicrobialagents, anti-imflammatory agents, heterologous or autologous growthfactors, growth factor fragments, small-molecule wound healingstimulants, proteins (including xenogeneic cartilage and matrixproteins), peptides, antibodies, enzymes, platelets, glycoproteins,hormones, glycosaminoglycans, nucleic acids, analgesics, viruses, virusparticles, and cell types. It is understood that one or more effectorsof the same or different functionality may be incorporated within theimplant.

Examples of suitable effectors include the multitude of heterologous orautologous growth factors known to promote healing and/or regenerationof injured or damaged tissue. These growth factors can be incorporateddirectly into the biocompatible scaffold, or alternatively, thebiocompatible scaffold can include a source of growth factors, such asfor example, platelets. Exemplary growth factors include, but are notlimited to, TGF-β, bone morphogenic protein, cartilage-derivedmorphogenic protein, fibroblast growth factor, platelet-derived growthfactor, vascular endothelial cell-derived growth factor (VEGF),epidermal growth factor, insulin-like growth factor, hepatocyte growthfactor, and fragments thereof. Suitable effectors likewise include theagonists and antagonists of the agents noted above. The growth factorcan also include combinations of the growth factors listed above. Inaddition, the growth factor can be autologous growth factor that issupplied by platelets in the blood. In this case, the growth factor fromplatelets will be an undefined cocktail of various growth factors.Platelets are normally found in the blood and play a role in hemostasisand wound healing. During clot formation, the platelets become activatedand release growth factors such as PDGF, TGF-β, VEGF, and IGF. Plateletscan be separated from blood using techniques such as centrifugation.When platelet rich plasma (PRP) is combined with an activator, aplatelet clot is created. An activator can be, but is not limited to,thrombin, calcium, adenosine di-phosphate (ADP), collagen, epinephrine,arachidonic acid, prostaglandin E2, ristocetin, calcium, retinoids,ascorbate, antioxidants, and combinations thereof.

The proteins that may be present within the implant include proteinsthat are secreted from a cell or other biological source, such as forexample, a platelet, which is housed within the implant, as well asthose that are present within the implant in an isolated form. Theisolated form of a protein typically is one that is about 55% or greaterin purity, i.e., isolated from other cellular proteins, molecules,debris, etc. More preferably, the isolated protein is one that is atleast 65% pure, and most preferably one that is at least about 75 to 95%pure. Notwithstanding the above, one of ordinary skill in the art willappreciate that proteins having a purity below about 55% are stillconsidered to be within the scope of this invention. As used herein, theterm “protein” embraces glycoproteins, lipoproteins, proteoglycans,peptides, and fragments thereof. Examples of proteins useful aseffectors include, but are not limited to, pleiotrophin, endothelin,tenascin, fibronectin, fibrinogen, vitronectin, V-CAM, I-CAM, N-CAM,selectin, cadherin, integrin, laminin, actin, myosin, collagen,microfilament, intermediate filament, antibody, elastin, fibrillin,tissue inhibitor of metalloproteinases (TIMPs), and fragments thereof.

Glycosaminoglycans, highly charged polysaccharides which play a role incellular adhesion, may also serve as effectors according to the presentinvention. Exemplary glycosaminoglycans useful as effectors include, butare not limited to, heparan sulfate, heparin, chondroitin sulfate,dermatan sulfate, keratan sulfate, hyaluronan (also known as hyaluronicacid), and combinations thereof.

The tissue repair implant of the present invention can also have cellsincorporated therein. Suitable cell types that can serve as effectorsaccording to this invention include, but are not limited to, osteocytes,osteoblasts, osteoclasts, fibroblasts, stem cells, pluripotent cells,chondrocyte progenitors, chondrocytes, endothelial cells, macrophages,leukocytes, adipocytes, monocytes, plasma cells, mast cells, umbilicalcord cells, stromal cells, mesenchymal stem cells, epithelial cells,myoblasts, tenocytes, ligament fibroblasts, neurons, and bone marrowcells. Cells typically have at their surface receptor molecules whichare responsive to a cognate ligand (e.g., a stimulator). A stimulator isa ligand which, when in contact with its cognate receptor, induces thecell possessing the receptor to produce a specific biological action.For example, in response to a stimulator (or ligand) a cell may producesignificant levels of secondary messengers, like Ca⁺², which then willhave subsequent effects upon cellular processes such as thephosphorylation of proteins, such as (keeping with our example) proteinkinase C. In some instances, once a cell is stimulated with the properstimulator, the cell secretes a cellular messenger usually in the formof a protein (including glycoproteins, proteoglycans, and lipoproteins).This cellular messenger can be an antibody (e.g., secreted from plasmacells), a hormone, (e.g., a paracrine, autocrine, or exocrine hormone),a cytokine, or natural or synthetic fragments thereof.

The tissue implant of the present invention can also be used in genetherapy techniques in which nucleic acids, viruses, or virus particlesdeliver a gene of interest, which encodes at least one gene product ofinterest, to specific cells or cell types. Accordingly, the biologicaleffector can be a nucleic acid (e.g., DNA, RNA, or an oligonucleotide),a virus, a virus particle, or a non-viral vector. The viruses and virusparticles may be, or may be derived from, DNA or RNA viruses. The geneproduct of interest is preferably selected from the group consisting ofproteins, polypeptides, interference ribonucleic acids (iRNA) andcombinations thereof.

Once the applicable nucleic acids and/or viral agents (i.e., viruses orviral particles) are incorporated into the tissue carrier matrix of thetissue repair implant, the implant can then be implanted into aparticular site to elicit a type of biological response. The nucleicacid or viral agent can then be taken up by the cells and any proteinsthat they encode can be produced locally by the cells. In oneembodiment, the nucleic acid or viral agent can be taken up by the cellswithin the tissue fragment of the minced tissue suspension, or, in analternative embodiment, the nucleic acid or viral agent can be taken upby the cells in the tissue surrounding the site of the injured tissue.One of ordinary skill in the art will recognize that the proteinproduced can be a protein of the type noted above, or a similar proteinthat facilitates an enhanced capacity of the tissue to heal an injury ora disease, combat an infection, or reduce an inflammatory response.Nucleic acids can also be used to block the expression of unwanted geneproduct that may impact negatively on a tissue repair process or othernormal biological processes. DNA, RNA and viral agents are often used toaccomplish such an expression blocking function, which is also known asgene expression knock out.

One skilled in the art will appreciate that the identity of thebiological component may be determined by a surgeon, based on principlesof medical science and the applicable treatment objectives.

In an exemplary method of forming the tissue repair implant of thepresent invention, the biocompatible, bioresorbable polymer or copolymermaterial is milled to form a powder. The particles of the resultantpowder, which serve as the granules of the implant, are then sorted bysize. The sorted granules are set aside, while a tissue samplecontaining viable cells is obtained. The tissue sample is minced,producing a fluid suspension containing the minced tissue fragments. Thefluid suspension is then introduced to a bed of the granules of aselected range of sizes. The bed of granules functions to sieve thefluid suspension, separating the fluid from the tissue fragments,resulting in a slurry or suspension containing both the granules and thetissue fragments. The slurry, which forms the tissue repair implant, canthen be collected and inserted into a delivery device such as aninjection device for immediate delivery to an injury or diseased tissuesite. The tissue fragments serve as a cell source for new cellulargrowth, and have an effective amount of viable cells that can migrateout of the tissue fragment to populate the tissue carrier matrix oncethe implant is delivered to the patient. The granules serve as amicrocarrier to provide sufficient mechanical integrity for cellularintegration with the surrounding environment during the tissueremodeling process. Because the implant is in a suspension form, theimplant can conform to any defect geometry and assume a shapecomplementary to that of the implantation site. The close interfacebetween the injected implant and the tissue site thus enhances tissuerepair and healing.

If desired, the tissue carrier matrix can also include a binding agentthat acts to gel together the tissue fragments and granules and therebyform a cohesive matrix. The binding agent can be added to the matrix toenable the implant to take on a gel-like or semi-solid form whichenhances retention of the implant at the tissue site while tissueremodeling is occurring. Where a solid or cured implant is desired, acuring agent can additionally be provided with the tissue carriermatrix. This curing agent would act to crosslink (i.e., form covalentbonds with) the binding agent, thereby forming a solid implant withinwhich are the tissue fragments and the bioresorbable, biocompatiblegranules. In one aspect, the curing agent is added to the implant afterdelivery to the implantation site so that the implant is set afterdelivery. In another aspect, the curing agent is added prior to deliveryof the implant to the site, resulting in a cured implant beingdelivered.

To further enhance the implant's regenerative or reconstructiveabilities, the tissue carrier matrix can also include a biologicalcomponent or effector which enhances the effectiveness of the tissuefragments to new cellular growth. In an exemplary method forincorporating the biological component, the tissue carrier matrix can beplaced in a suitable container comprising the biological component priorto surgical placement at the tissue site. After an appropriate time andunder suitable conditions, the granules can become impregnated with thebiological component. Following surgical placement, an implant in whichthe tissue carrier matrix is devoid of any biological component can beinfused with biological agent(s), or an implant in which the matrixincludes at least one biological component can be augmented with asupplemental quantity of the biological component. Another exemplarymethod of incorporating a biological component within a surgicallyinstalled implant is by injection using an appropriately gauged syringe.

The amount of the biological component included with the tissue repairimplant will vary depending on a variety of factors, including the sizeof the injury or defect site, the identity of the biological component,and the intended purpose of the tissue repair implant. One skilled inthe art can readily determine the appropriate quantity of biologicalcomponent to include within an implant for a given application in orderto facilitate and/or expedite the healing of tissue. The amount ofbiological component will, of course, vary depending upon the identityof the biological component and the given application.

It is also possible to add solids (e.g., barium sulfate) that willrender the tissue implants radio opaque. The solids that may be addedalso include those that will promote tissue regeneration or regrowth, aswell as those that act as buffers, reinforcing materials or porositymodifiers.

The tissue repair implant can be used in the treatment of a tissueinjury, such as injury to a ligament, tendon, nerve, skin, cartilage ormeniscus. Repairing tissue injuries involves the steps of obtaining asample of living tissue by any of the variety of techniques known tothose skilled in the art, preferably by biopsy or other minimallyinvasive techniques. The sample of living tissue is then processed understerile conditions to create at least one minced, finely divided tissueparticle, combining the tissue fragment with a plurality ofbiocompatible, bioresorbable granules to form a suspension of tissue andgranules, and injecting the suspension at a tissue injury site todeliver the tissue repair implant to the tissue injury. Additionally,the tissue repair implant can be allowed to set or cure into a shapecomplementary to the geometry of the defect site. In the alternative, itis contemplated that the suspension can also be introduced into a moldand allowed to set prior to implantation at the defect site. The moldcan have a geometry and dimension matching that of the defect site. Itis contemplated that a specialized surgical tool can be used to preparethe defect area so that the implantation site has a defined geometry.Once cured, the implant can be further trimmed and shaped as necessarybefore implantation.

In an exemplary method of repairing tissue using the implant of thepresent invention, a patient diagnosed with a symptomatic articularcartilage defect is prepped for arthroscopic surgery. The surgeon thenharvests healthy cartilage tissue from a non-weight bearing area of thepatient's joint using a harvesting instrument. Preferably, theinstrument also allows the surgeon to mince the cartilage tissue andcollect the minced tissue fragments in a separate chamber of theinstrument which is preloaded with the milled granules. Next, thesurgeon can inject a binding agent such as a biological glue into thechamber to prepare a mixture containing the cartilage fragments,granules and binding agent. A curing agent can also be introduced to thechamber at this time. While the mixture is being prepared, carefuldebridement of the affected area can be performed to remove unhealthytissue from the cartilage defect and prepare the area to receive themixture. The formed mixture can then be loaded into an injection devicesuch as a specialized syringe to arthroscopically inject the mixtureinto the affected area. After injection, the mixture can be shaped orsculpted to fill the defect area and the surrounding tissue before themixture has cured. Once cured, the arthroscopic ports can be suturedclosed, and the patient can then begin a controlled rehabilitationprogram.

The methods of repairing tissue injuries using the tissue implantsaccording to the present invention can be conducted during a surgicalprocedure to repair the tissue injury. Alternatively, the steps ofprocessing the tissue sample to create minced, finely divided tissueparticles, depositing the tissue particles upon the scaffold to form atissue repair implant, and/or incubating the tissue repair implant priorto implantation can be conducted at another, sterile location prior tosurgical placement of the implant relative to the site of injury.

The following example is illustrative of the principles and practice ofthis invention. Numerous additional embodiments within the scope andspirit of the invention will become apparent to those skilled in theart.

EXAMPLE

The primary objective of this study was to examine the outgrowth ofchondrocytes in a sample implant comprising bovine cartilage fragments,fibrin glue and polyglycolic acid (PGA) granules in vitro. For thestudy, minced bovine cartilage and PGA granules of different sizes weremixed with fibrin glue (Tisseel™) and injected into a mold to formbioadhesive plugs in accordance with Table 1 below. The resulting plugswere then cultured in cell culture or chondrogenic medium for 3 and 6week periods, respectively. After incubation, the plugs were fixed,sectioned and stained with hematoxylin and eosin (H & E).

TABLE 1 Experimental Conditions for Study of Chondrocyte Outgrowth fromComposite Mold Containing Fibrin Glue, PLA Granules, and BovineCartilage Fragments Amount of Incubation minced Amount of Amount ofperiod Conditions cartilage PGA fibrin glue 3 & 6 weeks Composite plug100 mg 31 mg 400 μl n = 1 each with PGA (158 μm) granules Composite plug100 mg 31 mg 400 μl n = 1 each with PGA (286 μm) granules Composite plug100 mg 31 mg 400 μl n = 1 each with PLA (633 μm) granules Fibrin glueplug 100 mg  0 mg 400 μl n = 1 each only

RESULTS

FIG. 1A shows a photomicrograph of a composite plug 10 with PGA granules12 of approximately 158 μm diameter and cartilage pieces 14 in fibringlue 16, made in accordance with the first condition of Table 1. FIG. 1Bshows a histological section of a composite plug in accordance with thefirst condition of Table 1, H & E stained, after 3 weeks. Slight cellgrowth is seen. After 6 weeks, however, the PGA granules show someresorption while the fibrin glue also indicates absorption and/ordegradation. As further shown in FIGS. 1C and 1D, which are histologicalsections of a composite plug made in accordance with the first conditionof Table 1, H & E stained, after 6 weeks, there appears to bechondrocyte outgrowth 18 around the cartilage fragments 14.

FIG. 2A shows a photomicrograph of a composite plug 20 with PGA granules22 of approximately 286 μm diameter and cartilage pieces 24 in fibringlue 26, made in accordance with the second condition of Table 1. FIG.2B shows a histological section of a composite plug in accordance withthe second condition of Table 1, H & E stained, after 3 weeks. Similarto FIG. 1B, slight cell growth is seen. After 6 weeks, however, the PGAgranules show some resorption while the fibrin glue also indicatesabsorption and/or degradation. As further shown in FIGS. 2C and 2D,which are histological sections, H & E stained, after 6 weeks of acomposite plug made in accordance with the second condition of Table 1,there appears to be chondrocyte outgrowth 28 around the cartilagefragments 24.

FIG. 3A shows a histological section of a composite plug 30 made inaccordance with the third condition of Table 1, containing cartilagefragments 34 and PLA granules 32 of approximately 633 μm diameterembedded in fibrin glue 36, stained with H & E after 3 weeks, at 100×magnification. FIG. 3B shows a histological section of the compositeplug 30 after 6 weeks. As shown, there is some outgrowth of chondrocytes38 around the granules 32 and glue 36 after 6 weeks.

Finally, FIGS. 4A and 4B show histological sections of a composite plug40 made in accordance with the fourth condition of Table 1, containingcartilage fragments 44 embedded in fibrin glue 46, stained with H & Eafter 3 weeks and 6 weeks, respectively, at 100× magnification. At 6weeks, FIG. 4B shows some cell outgrowth.

Based on the results of the study, it can be concluded that bovinechondrocytes from minced cartilage were able to migrate in and aroundthe fibrin glue bioadhesive plug within 6 weeks. It further appears thatthe presence of the polyglycolic acid (PGA) granules embedded within thefibrin glue plugs facilitated the migration process. Finally, it appearsthat the smaller sized PGA granules (mean particle diameter 158 μm and286 μm) were more effective in facilitating cell attachment and ininducing cellular migration to contribute to the overall tissueremodeling process.

One skilled in the art will appreciate further features and advantagesof the invention based on the above-described embodiments. Accordingly,the invention is not to be limited by what has been particularly shownand described, except as indicated by the appended claims. Allpublications and references cited herein are expressly incorporatedherein by reference in their entirety.

The invention claimed is:
 1. A tissue repair implant comprising: atissue carrier matrix in the form of an injectable suspension, thetissue carrier matrix comprising a plurality of biocompatible,bioresorbable granules and at least one tissue fragment in associationwith the tissue carrier matrix, the at least one tissue fragment havingan effective amount of viable cells that can migrate out of the tissuefragment and populate the tissue carrier matrix, and the granules havingan average outer diameter in a range of about 150 to about 300 μm. 2.The implant of claim 1, wherein the granules comprise at least one ofaliphatic polyesters, copoly(ether-esters), solid copolymers of fattyacid esters of glycerol and succinic acid, polyoxaesters, collagen,gelatin, albumin, hyaluronate, glycosaminoglycans, polyanhydrides,polysaccharides, polyphosphazines, subintestinal mucosa, and acellulartissues.
 3. The implant of claim 1, wherein the at least one tissuefragment has a particle size in the range of about 0.1 to about 2 mm³.4. The implant of claim 1, wherein the at least one tissue fragment isobtained from a connective tissue type selected from the groupconsisting of cartilage, meniscus, tendon, ligament, dermis, bone, fat,synovial tissue, muscle tissue, and combinations thereof.
 5. The implantof claim 1, wherein the tissue carrier matrix comprises at least one ofa proteoglycan, heparan sulfate, heparin, chondroitin sulfate, dermatansulfate, keratan sulfate, and hyaluronan.
 6. The implant of claim 1,wherein the at least one tissue fragment comprises a tissue selectedfrom the group consisting of autogenic tissue, allogeneic tissue,xenogeneic tissue, and combinations thereof.
 7. The implant of claim 1,wherein the granules comprise an aliphatic polyester selected from thegroup consisting of homopolymers or copolymers of lactides, glycolides,ε-caprolactone, p-dioxanone (1,4-dioxan-2-one), trimethylene carbonate(1,3-dioxan-2-one), and combinations thereof.
 8. The implant of claim 1,wherein the tissue carrier matrix further comprises at least onebiological component selected from the group consisting of antibiotics,antimicrobial agents, anti-inflammatory agents, growth factors, growthfactor fragments, small-molecule wound healing stimulants, hormones,cytokines, proteins, peptides, antibodies, enzymes, isolated cells,glycosaminoglycans, immunosuppressants, nucleic acids, analgesics,platelets, an activator of platelets, viruses, virus particles, andcombinations thereof.
 9. The implant of claim 8, wherein the at leastone biological component is an isolated cell selected from the groupconsisting of osteocytes, fibroblasts, stem cells, pluripotent cells,chondrocyte progenitors, chondrocytes, osteoclasts, osteoblasts,endothelial cells, macrophages, adipocytes, monocytes, plasma cells,mast cells, umbilical cord cells, leukocytes, stromal cells, mesenchymalstem cells, epithelial cells, myoblasts, tenocytes, ligamentfibroblasts, and bone marrow cells.
 10. A tissue repair implantcomprising: a tissue carrier in the form of an injectable suspension,the tissue carrier comprising a plurality of biocompatible,bioresorbable granules and at least one tissue fragment in associationwith the tissue carrier, the at least one tissue fragment having aneffective amount of viable cells that can migrate out of the tissuefragment and populate the tissue carrier, and the granules having anaverage outer diameter in a range of about 150 to about 300 μm.